Cl- transport by cystic fibrosis transmembrane conductance regulator (CFTR) contributes to the inhibition of epithelial Na+ channels (ENaCs) in Xenopus oocytes co-expressing CFTR and ENaC

Abstract

1. Epithelial Na+ channels (ENaCs) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is activated by protein kinase A. Since cAMP-dependent activation of CFTR Cl- conductance is defective in cystic fibrosis (CF), ENaC currents are not inhibited by CFTR. This could explain the enhanced Na+ conductance found in CF. In the present study, we examined possible mechanisms of interaction between CFTR and ENaC co-expressed in Xenopus oocytes. 2. The magnitude of CFTR Cl- currents activated by 3-isobutyl-1-methylxanthine (IBMX) in oocytes co-expressing either wild-type or mutant CFTR and ENaC determined the degree of downregulation of ENaC currents. 3. The ability of CFTR to inhibit ENaC currents was significantly reduced either when extracellular Cl- was replaced by poorly conductive anions, e.g. SCN- or gluconate, or when CFTR was inhibited by diphenylamine-carboxylate (DPC, 1 mmol l-1). 4. Downregulation of ENaC was more pronounced at positive when compared with negative clamp voltages. This suggests that outward currents, i.e. influx of Cl- through activated CFTR most effectively downregulated ENaC. 5. Activation of endogenous Ca2+-activated Cl- currents by 1 micromol l-1 ionomycin did not inhibit ENaC current. This suggests that inhibition of ENaC mediated by Cl- currents may be specific to CFTR. 6. The present findings indicate that downregulation of ENaC by CFTR is correlated to the ability of CFTR to conduct Cl-. The data have implications for how epithelia switch from NaCl absorption to NaCl secretion when CFTR is activated by secretagogues.

Figure 1. Representative examples of whole-cell currents observed in an oocyte co-expressing wild-type CFTR and ENaC

A, control (-IBMX); B, inhibition of ENaC by amiloride (10 μmol l⁻¹) in the absence of IBMX; C, stimulation of wtCFTR with 1 mmol l⁻¹ IBMX; D, inhibition of ENaC by amiloride (10 μmol l⁻¹) in the presence of IBMX. E and F, I–V curves corresponding to the experiments shown in A-D. Oocytes were voltage clamped in steps of 20 mV from -100 to +40 mV. Current values were measured 250 ms into each voltage step.

Figure 2. Inhibition of ENaC correlates with the magnitude of CFTR Cl⁻ conductance

A, amiloride (10 μmol l⁻¹)-sensitive ENaC conductance (ΔG_Amil) in ENaC (•) and ENaC + CFTR (○) expressing oocytes. Values for control and IBMX (1 mmol l⁻¹) are shown (n = 5). B, dependence of the inhibition of ENaC by the magnitude of whole-cell conductance activated during stimulation with IBMX. Residual ENaC conductance as a percentage is plotted vs. IBMX-activated whole-cell conductance (ΔG_IBMX). 100 % indicates no inhibition of ENaC and 0 % indicates that ENaC was completely inhibited through CFTR activated by IBMX (n = 57). Oocytes were voltage clamped in steps of 20 mV from -100 to +40 mV. Current values were measured 250 ms into each voltage step.

Figure 3. Inhibition of ENaC by wild-type and mutants of CFTR

Summary of the whole-cell conductances activated by stimulation with IBMX (1 mmol l⁻¹) (ΔG_IBMX, □). Amount of amiloride-sensitive ENaC conductance (amiloride 10 μmol l⁻¹) as a percentage of that seen under control conditions (-IBMX). 100 % indicates no inhibition of ENaC by CFTR, 0 % indicates complete inhibition of ENaC by CFTR. Oocytes were voltage clamped in steps of 20 mV from -100 to +40 mV. Current values were measured 250 ms into each voltage step. Values are means ±s.e.m. (number of experiments in parentheses).

Figure 4. Whole-cell currents observed in an oocyte co-expressing ENaC and wtCFTR

A, effects of amiloride (Amil, 10 μmol l⁻¹) on whole-cell currents were measured before and after stimulation of CFTR by IBMX (1 mmol l⁻¹). The oocyte was voltage clamped to either positive (+10 to +40 mV) or negative (-20 to -90 mV) voltages (see inset on left). B, summary of the experiments shown in A. Conductances were calculated separately for the negative and positive clamp voltage ranges (n = 8 paired experiments). * Statistically significant. C, concentration-response curves for the inhibition of ENaC by amiloride at positive and negative clamp voltages (n = 12).

Figure 5. Inhibition of ENaC by CFTR depends on the conducted anion

A, I–V curves obtained from oocytes expressing CFTR and ENaC after activation by IBMX (1 mmol l⁻¹). Effects of partial replacement of extracellular Cl⁻ (101 mmol l⁻¹) by gluconate (Gluc⁻) or SCN⁻ (both 96 mmol l⁻¹) on I–V curves. B, CFTR whole-cell Cl⁻ conductances were activated by 1 mmol l⁻¹ IBMX (IBMX vs. control) and were significantly (*) reduced when extracellular Cl⁻ was replaced subsequently by either gluconate (Gluc⁻, n = 8) or SCN⁻ (n = 8). Values are means ±s.e.m. (paired experiments). C, amiloride (10 μmol l⁻¹)-sensitive whole-cell conductance (ΔG_Amil) as measured for positive currents at the positive clamp voltage range (0 to +40 mV) in the absence of IBMX (control) and after stimulation with IBMX (1 mmol l⁻¹). Inhibition of ΔG_Amil by activation of CFTR in the presence of different extracellular anions is summarized (all paired experiments). ΔG_Amil is significantly inhibited by IBMX in the presence of either Cl⁻, gluc⁻ or SCN⁻ (asterisks). Inhibition of ΔG_Amil by CFTR is significantly reduced in the presence of either extracellular gluc⁻ or SCN⁻ compared with Cl⁻ (†). D, neither SCN⁻ nor Gluc⁻ had any significant effects on amiloride-sensitive ENaC conductance (ΔG_Amil) at either positive (V_c⁺) or negative (V_c⁺) clamp voltage (n = 3). Values are means ±s.e.m.

Figure 6. Inhibition of CFTR by DPC inhibits downregulation of ENaC by CFTR

A, inhibition of IBMX-activated (1 mmol l⁻¹) CFTR whole-cell Cl⁻ conductance by diphenylamine-carboxylate (DPC, 1 mmol l⁻¹, n = 7). B, inhibition of ENaC whole-cell conductance by amiloride (ΔG_Amil) was not influenced by DPC itself (n = 3, paired experiments). C, whole-cell currents observed in an oocyte co-expressing ENaC and wtCFTR. The effect of amiloride (Amil, 10 μmol l⁻¹) was examined under control conditions, after stimulation with IBMX (1 mmol l⁻¹) and in the presence of IBMX and DPC (1 mmol l⁻¹). D, amiloride-sensitive whole-cell conductances (ΔG_Amil) measured in oocytes co-expressing CFTR and ENaC. Inhibition of ΔG_Amil due to activation of CFTR by IBMX was attenuated in the presence of DPC (n = 4, paired experiments). Conductances were calculated for the voltage clamp range of -90 to +40 mV. Values are means ±s.e.m.* Significantly different from ΔG_Amil in the absence of IBMX; † significantly different from ΔG_Amil in the absence of DPC (all paired t tests).

Figure 7. Inhibition of ENaC by CFTR in the presence of low [Cl⁻]

A, inhibition of ENaC whole-cell conductance by amiloride (ΔG_Amil) in oocytes exposed to low (5 mmol l⁻¹, 5Cl) or high (101 mmol l⁻¹, 101Cl) extracellular Cl⁻ concentration (n = 5). B, whole-cell currents observed in an oocytes co-expressing ENaC and wtCFTR. Effects of amiloride (Amil, 10 μmol l⁻¹) in the presence or absence of IBMX (1 mmol l⁻¹) are shown when the extracellular Cl⁻ concentration was 5 mmol l⁻¹. C, summary of amiloride-sensitive whole-cell conductance (ΔG_Amil) calculated from experiments shown in B.ΔG_Amil was significantly (*) inhibited by IBMX in oocytes adapted to low extracellular (5 mmol l⁻¹) Cl⁻. Subsequent change to high extracellular Cl⁻ significantly (†) augmented inhibition of ΔG_Amil. Upon removal of IBMX in the presence of high extracellular Cl⁻, ΔG_Amil recovered from inhibition († significantly different from 101 Cl⁻+ IBMX; ² significantly different from 5 Cl⁻+ IBMX. Values are means ±s.e.m.

Figure 8. Ca²⁺-activated Cl⁻ conductance does not affect ENaC

A,I–V curves obtained from an oocyte exposed to 1 μmol l⁻¹ ionomycin (Iono). B, amiloride-inhibited (10 μmol l⁻¹) whole-cell ENaC conductance (ΔG_Amil) in the absence or presence of ionomycin (n = 5). C, effect of extracellular Ca²⁺ concentration (< 10⁻⁹ mol l⁻¹, -Ca²⁺; 1.5 mmol l⁻¹, +Ca²⁺) on IBMX-dependent inhibition of ΔG_Amil (n = 6) in oocytes co-expressing CFTR and ENaC. Values are means ±s.e.m., * significantly different.